The chemical synthesis of oligonucleotide refers to the process of connecting nucleotide units into an oligonucleotide chain by prompting the formation of 5′-3′ phosphate diester bonds between nucleotide monomers. It relates to the synthesis of protected nucleotides.
At present, the common synthesis process of oligonucleotide is solid-phase synthesis process: firstly, the 5′-OH on nucleotide is protected by 4,4′-dimethoxytrityl (DMT), the amino on the base is protected by benzoyl, and the 3′-OH is activated by amino phosphite compound. The 3′-OH on the first nucleotide is combined with solid-phase resin, the protective group on 5′-OH is removed, a phosphite triester is formed between the exposed 5′-OH and the 3′-OH of the second nucleotide, which is activated by amino phosphite compound, the phosphite triester forms phosphotriester through iodination, and then trichloroacetic acid is added to remove the protective group on the 5′-OH of the second nucleotide. By now, the oligonucleotide chain is extended by one nucleotide unit, and may be put into the next round of extension reaction. After the synthesis of the whole oligonucleotide segment is completed through several rounds of extension reaction, concentrated ammonium hydroxide is used to remove the oligonucleotide segment from the solid-phase resin, and after deprotection and purification, oligonucleotide is obtained.
The advantages of the said solid-phase synthesis process lie in: 1) automation: all the synthetic reactions are automatically completed by a synthesizer; 2) short synthetic cycle; 3) high yield: the yield of single-step condensation reaction is greater than 98% in general. However, the solid-phase synthesis process also has some defects: 1) small synthetic scale: the scale of solid-phase synthesis does not exceed 100 μmol in general, which is far from meeting the requirement of the use in pharmaceutical raw materials; 2) difficulty in achieving high purity: due to the limitation of the synthesis process, it is inevitable that the obtained oligonucleotide contains non-target oligonucleotide segments with N−1, N−2 of base number, which are rather detrimental to pharmaceutical application; 3) serious waste: to realize sufficient reaction, in each synthetic cycle, often phosphoramidite monomers that are several times as much as the amount actually consumed by the reaction need to be added to realize excess, and after the cycle, a large amount of organic solvent has to be used as a washing solvent to wash away the unreacted phosphoramidite monomers; 4) high cost: the solid-phase carrier and phosphoramidite monomers needed by the reaction are expensive, resulting in high cost of oligonucleotide synthesis.
Due to the important function of oligonucleotide in life activities and the fast development of nucleic acid research technology, particularly the development of RNA interference technology and its potential clinical application value, large-scale synthesis of oligonucleotide is a matter of significance.